Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

Abstract

Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK). Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-'addicted' human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases.

Figure 1. RTK ligands attenuate kinase inhibition in oncogene-addicted cancer cell lines

a, Summary of results from 41 kinase-addicted cancer cell lines co-treated with the appropriate kinase inhibitor and each of six RTK ligands. NR, no rescue; P, partial rescue; R, complete rescue. b, Cell viability assay demonstrating the diversity of ligand effects on drug-treated cell lines (72 h), illustrating examples of no rescue, partial rescue or complete rescue. Lap, lapatinib. Graphs show average values of technical duplicates from one representative experiment out of three independent experiments.

Figure 2. Pro-survival pathway re-activation correlates with RTK-ligand rescue

a, Immunoblots showing effects of ligand (50 ng ml⁻¹) on RTK, AKT and extracellular signal-regulated kinase (ERK) phosphorylation (p) after kinase inhibition (1 μM, 2 h). Ligand rescue is indicated: green squares, complete rescue; blue squares, partial rescue. b, Drug-induced suppression of viability in three kinase-addicted cell lines (72 h). Cells were co-treated with ligand and an appropriate secondary kinase inhibitor (0.5 μM). Graphs show average values of technical duplicates from one representative experiment out of three independent experiments. Criz; crizotinib; Erl, erlotinib; Lap, lapatinib; PD, PD173074; PLX, PLX4032; Sun, sunitinib. c, Immunoblots showing effect of kinase inhibition (1 μM) ± ligands (2 h) on RTK, AKT and ERK phosphorylation. Cells were co-treated with secondary kinase inhibitor (0.5 μM).

Figure 3. HGF promotes lapatinib resistance in HER2-amplified breast cancer cell lines

a, Immunoblots showing apoptosis in AU565 cells after lapatanib (Lap; 1 μM), HGF or crizotinib (Criz; 0.5 μM) treatment. Blue font indicates partially rescued cell lines. b, Immunoblots showing phospho-MET (pMET) and MET in HER2-amplified breast cancer cell lines with partial HGF rescue indicated. c, Syto 60 cell staining of AU565 cells treated with lapatinib (1 μM), HGF or crizotinib (0.5 μM). d, Immunoblots showing AKT and ERK re-activation in MET-positive (blue) and MET-negative (black) cells. Cells were treated with lapatinib (1 μM), HGF or crizotinib (0.5 μM) (2 h). e, Syto 60 staining of HCC1954 cells treated with lapatinib (5 μM) or crizotinib (1 μM). Images are representative of three biological replicates and values indicate mean ± s.d.

Figure 4. HGF promotes PLX4032 resistance in BRAF-mutant melanoma cell lines

a, Left, immunoblots showing MET status in melanoma cells, with HGF rescue indicated. Right, correlation between MET expression and HGF rescue in PLX4032 (1 μM)-treated cells (72 h). Black font indicates no rescue; blue, partial rescue; green, complete rescue. b, Immunoblots showing ERK reactivation in MET-positive (green/blue) and MET-negative (black) cells treated with PLX4032 (1 μM), HGF or crizotinib (0.5 μM) (2 h). c, Effect of activating MET (3D6) on tumour growth inhibition by PLX4032 in two xenografts (n =10 per group). mAb, monoclonal antibody; PLX, PLX4032. Differences between PLX4032-treated and PLX4032- and GDC-0712 (MET inhibitor)-treated control antibody (gp120) groups (*P =0.0008). Error bars represent mean ± s.e.m. (biological replicates). d, e, PFS (d) and OS (e) in PLX4032-treated melanoma patients stratified based on plasma HGF (green < median HGF; red > median HGF).